The location of central respiratory chemoreceptors and their cellular mechanisms have not been defined. Understanding these mechanisms is important because blood CO2 level is the dominant drive for respiration under normal conditions, and because abnormalities of chemoreception occur in many common diseases, including chronic obstructive pulmonary disease, sleep apnea and possibly sudden infant death syndrome. Previous work in this laboratory has identified putative central respiratory chemoreceptor neurons within the medullary raphe, which are highly chemosensitive. These neurons have now been isolated in tissue culture, where they express the same electrophysiological properties, chemosensitivity and neurotransmitters as medullary raphe neurons in brain slices and in vivo. Together with ongoing work in brain slices, this tissue culture model of cellular chemosensitivity provides an opportunity to study the fundamental mechanisms of central respiratory chemoreception at a highly detailed cellular level. The proposed work will examine a number of basic unanswered questions about chemosensitivity of medullary neurons. 1) How does the degree of chemosensitivity of medullary raphe neurons compare to that of neurons within the ventrolateral medulla and other brainstem respiratory nuclei that are also putative sites for chemoreception? 2) What are the neurotransmitters contained in chemosensitive medullary neurons? 3) How does acetylcholine modulate chemosensitive neurons? 4) Does CO2 act through a change in intracellular pH alone, or does extracellular pH or CO2 also modulate neuronal activity? 5) What are the ionic mechanisms of chemosensitivity? One set of experiments will use perforated patch clamp recordings to quantitatively compare chemosensitivity of neurons within the medullary raphe with other putative chemoreceptor sites. Another set of experiments will first identify chemosensitive neurons of the medullary raphe, then immunohistochemistry will be used to determine which neurotransmitters they contain. A third set of experiments will use patch clamp recordings to determine how chemosensitive raphe neurons are affected by acetylcholine agonists. A combination of patch clamp recording and fluorescence measurement of intracellular pH will then be used to determine the primary stimulus to chemosensitive raphe neurons. Preliminary work has identified a novel calcium-activated nonselective cation current in stimulated raphe neurons. Whole-cell patch clamp recordings will be used to determine the properties of this current, and how respiratory acidosis modulates it. For the first set of experiments, brain slices will be used exclusively. For the remaining experiments, cultured neurons will be used along with brain slices to verify the observations seen in culture. The proposed experiments should define the neurotransmitter content, and mechanisms of chemosensitivity of the medullary raphe, a brainstem site that is an excellent candidate for central respiratory chemoreception. Disturbances of breathing are common in human diseases. Understanding the basic mechanisms involved in modulation of neuronal activity by CO2 and pH may help provide successful treatment for these diseases.